RNA polymerase is the central enzyme of gene expression in all domains of life. Changes in gene expression patterns punctuate the progress of normal and neoplastic development of eukaryotes and onset of virulence in bacteria. Elongation is the longest and a highly regulated part of transcription cycle. Elongation factors such as PTEF-b and RfaH are implicated in development of cancer in humans and virulence in bacteria, respectively. This project aims at elucidation of the detailed molecular mechanism of transcript elongation by bacterial RNA polymerase by combining the high-resolution structural analysis of elongation intermediates with the state-of-the-art biochemical characterization of the elongation mechanism. Different states of the transcription complex that have been detected by biochemical and single-molecule experiments likely serve as targets for protein factors, small RNAs, nucleotide analogs, antibiotics, and other regulators of gene expression. Structures of RNA polymerase complexes that represent functional intermediates in the transcription cycle will be determined for the two model bacterial systems, Escherichia coli and Thermus thermophilus. The former is the major source of the biochemical and genetic data, whereas the latter afforded the high-resolution RNAP structures. Specific aims of this study will be as follows. First, to determine organization and structure of the transcription elongation complex at the atomic level. Nucleic acid scaffolds will be used to assemble, characterize, and crystallize functional elongation complexes captured in different conformational states and to solve their structures. Structure-based site-directed mutagenesis will be then applied to examine functional importance of the key elements of the transcription elongation complex. Second, to elucidate the mechanism of substrate selection and catalysis by bacterial RNA polymerase, structures of the transcription elongation complexes with the incoming substrates will be determined by X-ray analysis. Subsequent functional tests will be designed to identify the determinants for nucleotide selection and incorporation; in parallel, genetic approaches will be used to study the regulation of transcriptional fidelity in vivo. Third, to elucidate the elongation complex determinants that control both its high thermodynamic stability and the facile translocation upon nucleotide addition, the effects of systematic variation of the scaffold components and substitutions of the key RNA polymerase residues that are highlighted by the structure will be analyzed